The goal of this project is to characterize and control novel epigenetic regulatory mechanisms that drive altered gene activity in cancer. The majority of our initial effort in this area has focused on the study of protein and nucleic acid acetylation. Using a suite of novel chemical tools that allow us to study acetylation mechanisms directly in cells for the first time, we have discovered new enzymatic and non-enzymatic acetylation pathways that are highly elevated in cancer. We have also made substantial progress characterizing the activity, druggability, and metabolic regulation of these mechanisms. These advances are grouped according to four specific aims. 1. Discovery and characterization of novel acetyltransferase enzymes. Chemoproteomic profiling methods pioneered in our lab led to the discovery of a relatively uncharacterized acetyltransferase, NAT10, which is highly upregulated in a variety of cancer cell lines and also sensitive to the metabolic state of the cell. Subsequent work has revealed the primary function of NAT10 is the catalysis of RNA cytidine acetylation. To understand the role of dynamic NAT10 activity in cancer, we have developed first-in-class methods to study cytidine acetylation towards the goal of defining its role in acetylation-dependent gene regulation and cell growth. In addition to this work, we have continued to extend our chemoproteomic profiling methods to biological systems of increasing complexity in order to determine new functions of enzymatic and non-enzymatic acetylation mechanisms in cellular homeostasis, differentiation, and disease. 2. Characterization of acetyltransferase inhibitors. Targeting the cellular acetylation machinery is an emerging paradigm in oncology. However, relatively few small molecule inhibitors of acetyltransferases are known. To address this unmet need, our group has developed biochemical, chemoproteomic, and cell-based assays that can be used to unambiguously interrogate the activity of small molecule acetyltransferase inhibitors. These methods enabled the first evidence for cellular occupancy and on-target activity of a small molecule lysine acetyltransferase inhibitor. Currently we are applying these approaches towards the discovery of novel acetyltransferase inhibitors. Another important application of this work has been the identification of pan-assay interference features of reported acetyltransferase inhibitors, which has aided the interpretation of the activity of these molecules in cellular assays. 3. Metabolic regulation of epigenetics. We have previously applied our chemoproteomic profiling methods to define short- and long chain fatty acyl-CoAs as novel endogenous inhibitors of acetyltransferase activity. In addition, we have used a covalent chemoproteomic strategy to identify lysine succinylation and malonylation as markers of functional non-enzymatic acylation in the mitochondria and nucleus, respectively. Our current studies are focused on understanding the role of reactive acyl-CoAs in gene regulation, identifying novel classes of reactivity-guided acylations, and extending our chemoproteomic profliling methods to gain novel insights into other covalent metabolites, including the oncometabolite fumarate. 4. Diagnostic detection of oncometabolites. Recently, we reported a fluorogenic reaction that could be used to detect fumarate, an oncometabolite that accumulates to high levels in the hereditary kidney cancer predisposition syndrome HLRCC. In the past year we have identified novel chemical scaffolds that greatly improve the sensitivity of this approach and also allow it to be applied directly in living cells. We are currently applying this approach to identify novel drivers of elevated fumarate in cancer, and small molecules that may be helpful in restoring this balance. These combined aims leverage new cellular enzyme profiling technologies to discover novel epigenetic mechanisms, validate next-generation therapeutics, and develop novel diagnostics for cancer treatment.